TY - BOOK

T1 - Creation of hypoallergenic mustard (Brassica juncea) through genome editing and development of precise base editing tools for plants

AU - Zhang, Dingbo

PY - 2023

Y1 - 2023

N2 - Improved agricultural safety through novel breeding techniques is urgently required to increase access to nutritious foods worldwide. Genome engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-based or transcription activator-like effector (TALE)-based technologies provides a unique ability to modify targeted genes for precise breeding. This technology shows promise in various applications of allergy research. The major allergen Bra j I from brown mustard (Brassica juncea) is a seed storage protein that belongs to the 2S albumin family. One aim of this thesis was to create a hypoallergenic variety of mustard by utilizing genome editing techniques and a second aim was to develop novel base editing tools for plants. Firstly, two CRISPR/Cas9 constructs with multiplex single guide RNAs were employed to induce large deletions or frameshift mutations in both Bra j IA and Bra j IB homoeologs in two brown mustard lines (Terratop and CR2664). High mutation efficiencies were observed in the T0 transgenic mustard plants. The Bra j IB allele exhibited large deletions ranging from 566 to 790 bp in four lines. Additionally, nine out of 18 Terratop T0 lines exhibited small indels in the targeted regions. Similarly, 14 out of 16 CR2664 T0 lines analyzed had indels, while three lines exhibited mutations in all four Bra j I alleles. The mutations were stably inherited to the T1 progeny. Moreover, immunoblotting results demonstrated a decrease or complete absence of the Bra j I protein in the seed extracts of selected T1 lines. This work highlights the value of genome editing technologies in creating hypoallergenic food plants. Secondly, two base editing tools: TALE-derived DddA-based cytosine base editors (TALEDdCBEs) and TALE-derived adenine base editors (TALE-ABEs) were developed for precise C•G-to-T•A and A•T-to-G•C editing, respectively. TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice protoplasts. TALE-DdCBEs containing DddA11 exhibited broader sequence compatibility for editing non-TC targets. Furthermore, a series of TALE-ABEs with different deaminase fusion architectures were tested in N. benthamiana and rice. The results showed that TALE-ABEs enable the conversion of A•T-to-G•C in rice protoplast. The application of TALE-base editors can result in a dramatic change because they can be deployed for nuclear genes or, alternatively, target the genomes of plastids or mitochondria by N-terminal targeting sequences.

AB - Improved agricultural safety through novel breeding techniques is urgently required to increase access to nutritious foods worldwide. Genome engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-based or transcription activator-like effector (TALE)-based technologies provides a unique ability to modify targeted genes for precise breeding. This technology shows promise in various applications of allergy research. The major allergen Bra j I from brown mustard (Brassica juncea) is a seed storage protein that belongs to the 2S albumin family. One aim of this thesis was to create a hypoallergenic variety of mustard by utilizing genome editing techniques and a second aim was to develop novel base editing tools for plants. Firstly, two CRISPR/Cas9 constructs with multiplex single guide RNAs were employed to induce large deletions or frameshift mutations in both Bra j IA and Bra j IB homoeologs in two brown mustard lines (Terratop and CR2664). High mutation efficiencies were observed in the T0 transgenic mustard plants. The Bra j IB allele exhibited large deletions ranging from 566 to 790 bp in four lines. Additionally, nine out of 18 Terratop T0 lines exhibited small indels in the targeted regions. Similarly, 14 out of 16 CR2664 T0 lines analyzed had indels, while three lines exhibited mutations in all four Bra j I alleles. The mutations were stably inherited to the T1 progeny. Moreover, immunoblotting results demonstrated a decrease or complete absence of the Bra j I protein in the seed extracts of selected T1 lines. This work highlights the value of genome editing technologies in creating hypoallergenic food plants. Secondly, two base editing tools: TALE-derived DddA-based cytosine base editors (TALEDdCBEs) and TALE-derived adenine base editors (TALE-ABEs) were developed for precise C•G-to-T•A and A•T-to-G•C editing, respectively. TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice protoplasts. TALE-DdCBEs containing DddA11 exhibited broader sequence compatibility for editing non-TC targets. Furthermore, a series of TALE-ABEs with different deaminase fusion architectures were tested in N. benthamiana and rice. The results showed that TALE-ABEs enable the conversion of A•T-to-G•C in rice protoplast. The application of TALE-base editors can result in a dramatic change because they can be deployed for nuclear genes or, alternatively, target the genomes of plastids or mitochondria by N-terminal targeting sequences.

U2 - 10.15488/14747

DO - 10.15488/14747

M3 - Doctoral thesis

CY - Hannover

ER -